Developmental Cell

Article

Distinct Regulatory Cascades Govern Extraocularand Pharyngeal Arch Muscle Progenitor Cell FatesRamkumar Sambasivan,1,2 Barbara Gayraud-Morel,1,2 Gerard Dumas,1,2 Clemire Cimper,2 Sylvain Paisant,1,2

Robert Kelly,3 and Shahragim Tajbakhsh1,2,*1Institut Pasteur, Stem Cells & Development, CNRS URA 25782Department of Developmental Biology, 25 rue du Dr. Roux, 75724 Paris Cedex 153Developmental Biology Institute of Marseilles-Luminy, UMR 6216 CNRS

Universite de la Mediterranee, Campus de Luminy, Case 907, 13288 Marseille Cedex 9, France

*Correspondence: shaht@pasteur.fr

DOI 10.1016/j.devcel.2009.05.008

SUMMARY

Genetic regulatory networks governing skeletal myo-genesis in the body are well understood, yet theirhierarchical relationships in the head remain unre-solved. We show that either Myf5 or Mrf4 is neces-sary for initiating extraocular myogenesis. WhereasMrf4 is dispensable for pharyngeal muscle progen-itor fate, Tbx1 and Myf5 act synergistically for gov-erning myogenesis in this location. As in the body,Myod acts epistatically to the initiating cascades inthe head. Thus, complementary pathways, governedby Pax3 for body, and Tbx1 for pharyngeal muscles,but absent for extraocular muscles, activate the coremyogenic network. These diverse muscle progeni-tors maintain their respective embryonic regulatorysignatures in the adult. However, these signaturesare not sufficient to ensure the specific muscle pheno-types, since the expected differentiated phenotypeis not manifested when satellite cells are engraftedheterotopically. These findings identify novel geneticnetworks that may provide insights into myopathieswhich often affect only subsets of muscles.

INTRODUCTION

During development, instructive signaling and positional cues

impinge on gene regulatory networks that act in a hierarchical

fashion for the specification, commitment, and differentiation

of stem and progenitor cells. Transcriptional cascades that

govern lineage commitment are crucial for orchestrating organ-

ogenesis and development. Muscle stem cell self-renewal and

commitment are controlled by a genetic network including the

paired-box transcription factors Pax3 and Pax7, and basic

helix-loop-helix muscle regulatory factors (MRFs) Myf5, Mrf4,

Myod, and Myogenin (Kassar-Duchossoy et al., 2004; Relaix

et al., 2005; Rudnicki et al., 1993; Seale et al., 2000; Tajbakhsh

et al., 1997). Genes in the core regulatory network comprising

Myf5, Mrf4, and Myod act as the obligate determinants for di-

recting myogenic cell fate (Kassar-Duchossoy et al., 2004; Rud-

nicki et al., 1993), whereas Myogenin functions as an essential

differentiation factor (Hasty et al., 1993; Nabeshima et al., 1993).

810 Developmental Cell 16, 810–821, June 16, 2009 ª2009 Elsevier

The genetic linkage between Myf5 and Mrf4 had obscured the

role of Mrf4 in earlier studies (Braun and Arnold, 1995; Olson

et al., 1996; Rudnicki et al., 1993; Tajbakhsh et al., 1996,

1997). Using an allelic series of Myf5 mutants that differentially

affect Mrf4 expression, it was shown that Mrf4 and Myf5 act

upstream of Myod in the myogenic program of somites (Kas-

sar-Duchossoy et al., 2004). Myogenesis in the head is less

well investigated (Grifone and Kelly, 2007), and it is generally

thought that Myf5 and Myod act redundantly and in parallel for

initiating head muscle cell fate, while the role of Mrf4 is unknown.

In vertebrates, muscle progenitor cell (MPC) specification

occurs first in somites (Sambasivan and Tajbakhsh, 2007) and

these MPCs make body muscles. Notably, MPCs in the head

are derived from cranial mesoderm (Noden and Francis-West,

2006), and some signaling cascades act distinctly in somitic

and cranial mesoderm (Hacker and Guthrie, 1998; Mootoosamy

et al., 2002; Tzahor et al., 2003). Whereas extraocular muscles

(EOMs) develop in the periocular region, muscles of the face,

jaw, and neck develop from progenitor cells in the mesoderm

of the pharyngeal arches (PAs). EOMs govern eye movements

and have unique properties including expression of specific

Myosin heavy-chain isoforms (Porter, 2002; Sartore et al., 1987).

Pax3 and Pax7 play critical roles in somitic progenitors (Sam-

basivan and Tajbakhsh, 2007). Notably, Pax3:Pax7 double-

mutant somites are severely hypoplastic (Relaix et al., 2005)

where muscle stem and progenitor cells are eventually lost by

apoptosis; therefore, these genes regulate cell survival as well

(see Sambasivan and Tajbakhsh, 2007). Remarkably, Pax3:

Myf5(Mrf4) mutants (Mrf4 compromised in cis) uncouple myo-

genesis in the head from that in the body, where in the latter,

muscles do not form due to a failure to activate Myod (Tajbakhsh

et al., 1997). Therefore, Pax3 acts in a genetic pathway parallel to

Myf5 and Mrf4, and rescues Myod-mediated myogenesis in their

absence.

Although these regulatory networks have been largely estab-

lished for somite derived muscles, the hierarchical interactions

conferring muscle cell fate in the head have been elusive (Grifone

and Kelly, 2007; Sambasivan and Tajbakhsh, 2007). Resolving

these genetic relationships is critical since different myopathies

affect only subgroups of muscles in the body or head (Emery,

2002), yet the underlying mechanism of these clinical observa-

tions remains unresolved. Given that overall head muscle devel-

opment appears to be unhindered in Pax3:Myf5(Mrf4) mutants

and that Pax3 is not expressed in head MPCs or muscles,

Inc.

Developmental Cell

Distinct Regulatory Cascades Govern Head Myogenesis

Pax7, which is expressed in adult head progenitors (Horst et al.,

2006; Nathan et al., 2008), or other transcriptional regulators,

may cooperate with the core MRF network in this location. The

sine oculus (Six) and eyes absent (Eya) genes are key regulators

as Six1:Six4 and Eya1:Eya2 double nulls are deficient in limb

MPCs (Grifone et al., 2005, 2007). However, they do not rescue

Myod activation in Pax3:Myf5(Mrf4) mutants (Tajbakhsh et al.,

1997), suggesting that they act outside the core (MRFs) and

complementary (Pax3) network.

Interestingly, Musculin (Msc or MyoR), Tcf21 (Capsulin), Pitx2,

and Tbx1 play important roles in head muscle development (Gri-

fone and Kelly, 2007; Noden and Francis-West, 2006). Embryos

double null for the transcriptional repressors Msc and Tcf21 fail

to develop a subset of first-arch-derived muscles (Lu et al.,

2002). The bicoid-related homeodomain gene Pitx2 and the T-

box containing gene Tbx1 are expressed widely in the devel-

oping head, as well as in the body (Chapman et al., 1996; Kita-

mura et al., 1999; L’Honore et al., 2007). In Pitx2 null mice,

first-arch-derived muscles do not develop normally (Dong

et al., 2006; Shih et al., 2007), EOMs are lost (Gage et al.,

1999; Kitamura et al., 1999), and their progenitors mislocalized

(Dong et al., 2006). In Tbx1 mutants, myogenesis in the first

arch is impaired and muscles derived from other arches are

absent due primarily to a loss of these structures (Jerome and

Papaioannou, 2001; Kelly et al., 2004). A random complement

of hypomorphic first-arch-derived muscles are observed at later

stages in Tbx1 null embryos, revealing a requirement for Tbx1 in

ensuring robust bilateral activation of the myogenic program in

the mandibular arch (Kelly et al., 2004). Therefore, a complex

circuitry governs head myogenesis (PA versus EOM) and the

genetic relationships between these regulators, and with the

core MRFs, remain unresolved.

Using genetic manipulations and cell transplantations in mice,

we show that head and body myogenic regulatory cascades

have co-opted different combinations of a core and complemen-

tary pathway. Unlike all other skeletal muscles in the embryo,

EOMs are entirely dependent on Myf5/Mrf4. We show also

that, like Pax3 in the body, Tbx1 operates complementary to

Myf5 and acts genetically upstream of Myod in the PA. Interest-

ingly, the molecular signature of adult progenitors reflects their

developmental history, yet heterotopically transplantated adult

head satellite cells do not retain their original phenotype upon

differentiation. We propose that multiple distinct genetic

programs regulate overall skeletal muscle development in the

mouse.

RESULTS

An Obligate Requirement for Myf5 or Mrf4 to InitiateEOM Cell FateTo investigate developmental myogenesis in the vertebrate

head, we focused first on the patterning of EOMs in mice. How

MPCs are born, migrate, and pattern the EOMs is unknown.

Myf5nlacZ/+ and Myf5nlacZ/nlacZ embryos of different stages were

stained using X-gal for whole mount visualization of EOMs. A

single bilateral EOM anlage which is apparent at E10.5 (see

Figure S1A available online), appears to initiate the other EOMs

after splitting and patterning as evidenced at E11.5 and E12.5

(Figures 1A, 1C, 1E, S1C, S1D, and S1E). By E13.5 all EOMs

Deve

are properly positioned (data not shown). In Myf5nlacZ/nlacZ

embryos (Mrf4 also inactivated in cis; Kassar-Duchossoy et al.,

2004), the initial EOM anlage was formed and expanded in the

mutants, but surprisingly, it failed to pattern and began to

atrophy by E11.5 (Figures 1B and 1D; Figure S1B). This initial

growth, followed by a decline, indicates that the MPCs are

born but are eventually lost in these mutants. This is not the

case for any of the other MPCs (somites, limbs, pharyngeal

arches; Tajbakhsh et al., 1996, 1997; data not shown). Staining

with antibodies against activated Caspase-3 and TUNUL

labeling, which mark apoptotic cells, revealed that the mutant

anlagen had 5–6-fold more apoptotic cells compared to the

control (Figures 1F and 1H–1I0, and data not shown). Costaining

with an anti-b-galactosidase antibody to demarcate this struc-

ture and Ki67 or Cyclin D1 to identify cycling cells, showed no

significant difference in the proliferation of mutant progenitors

(Figures 1G and 1J–1K0; data not shown). Thus, disruption of

EOM patterning as well as loss of EOM progenitors by apoptosis

occurs in Myf5nlacZ/nlacZ mutants.

Mrf4 is expressed in EOM progenitors (Figures S1F–S1H00) and

since the Myf5nlacZ allele results in compromised Mrf4 expres-

sion, we wondered if the EOM defects in Myf5nlacZ/nlacZ mutants

were due to the combined loss of these genes. To address this

point, we examined an allelic series of Myf5 knockins which

progressively affect Mrf4 expression in MPCs: Myf5loxP/loxP (not

affected significantly), Myf5GFP-P/GFP-P (�50% reduced), and

Myf5nlacZ/nlacZ (fully compromised) (Kassar-Duchossoy et al.,

2004). At E14.5, a stereotyped EOM pattern is normally observed

by immunostaining for Myosin Heavy chain (MyHC; Figures 2A–

2C). Notably, we found a range of EOM phenotypes correspond-

ing to the extent of disruption of Mrf4 expression among the

Myf5 null alleles (Figures 2D–2F). In Myf5loxP/loxP mutants, all

anlage of EOMs were present, although some were hypoplastic

(Figure 2D). Myf5GFP-P/GFP-P mutants displayed an intermediate

phenotype, where some EOMs were severely reduced, and

others were missing (Figure 2E). Notably, Myf5nlacZ/nlacZ embryos

completely lacked all EOMs or had only residual myofibers at

birth (Figure 2F; data not shown). Accordingly, Mrf4nlacZ-P/nlacZ-P:

Myf5loxP/loxP mutants where both Mrf4 and Myf5 are inactivated

by gene targeting (Kassar-Duchossoy et al., 2004) phenocopied

the Myf5nlacZ/nlacZ mutant (Figure 2G).

To determine if Mrf4 is necessary for EOM development we

examined Mrf4�/� null mutants (Zhang et al., 1995). These

embryos have mild EOM defects (Figure 2H), comparable in

severity to Myf5loxP/loxP. To assess if delayed activation of

Myod or Myogenin eventually rescues extraocular myogenesis,

we dissected out eyeballs from adult mutants along with the

retinue of EOMs and immunostained them in whole-mount with

anti-MyHC antibody. As shown in Figures 2J–2L, newborn or

adult mutants display the defects that reflect those observed in

the early fetus. Thus, we conclude that either Myf5 or Mrf4 is

necessary for EOM progenitors to acquire their myogenic fate,

and unlike muscles elsewhere, Myod does not rescue EOM

development.

Lineage Progression and Cell Survival Are Distinctin EOM and Somitic MPCsWe reported previously that somitic MPCs are developmentally

arrested along the dermomyotome (DM) epithelium for about

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Developmental Cell

Distinct Regulatory Cascades Govern Head Myogenesis

A

G

B

C

J’ K’

D

J

H’ I’

E

KH I

F Figure 1. Patterning and Fate of Extraocular

Muscle Progenitors

(A and C) X-gal stained E11.5, E12.5 Myf5nlacZ/+

embryos, rendered transparent by benzyl

alcohol-benzyl benzoate (BABB) treatment. (C

and D) Myf5nlacZ/nlacZ embryos staged and treated

as in (A and C). Mrf4 expression is compromised in

cis in Myf5nlacZ allele. White arrowheads point to

emerging pattern in controls (A and C) and loss

of MPCs in null mutants (B and D). White arrows

mark EOM anlagen delineated by white boundary;

black arrows indicate PA-derived muscles.

(E) Box indicates the region shown in (A–D).

(F and G) Quantifications of apoptotic ([F] from

[H–I0]; mutants 43.1% ± 5.8 standard error of the

mean [SEM], 140 cells total; control 7.8% ± 1.3

SEM, 250 cells total; **p value 0.0026; Student’s t

test; n = 4 embryos each) and cycling ([G], [J–K0])

cells among EOM progenitors in Myf5 null mutants

in comparison to heterozygous control, normalized

to (F) or expressed as a percentage of (G) Myf5nlacZ

and Myf5GFP-P (data not shown) reporter positive

cells, respectively.

(H–I0) Sagittal sections of heads of E11 Myf5nlacZ/+

(H and H0 ) and Myf5nlacZ/nlacZ (I and I0)

embryos immunostained with anti-b-gal and anti-

Caspase3activated, a marker for apoptotic cells.

Inset in (I0) shows a b-gal+ apoptotic cell.

(J–K0 ) Sagittal sections of heads of E11.5 Myf5nlacZ/+

(J and J0 ) and Myf5nlacZ/nlacZ (K and K0) embryos

immunostained with anti-b-gal and anti-Cyclin D1.

TV, telencephalic vesicle; 1st PA, 1st pharyngeal

arch. Scale bars: 50 mm in ([J–K0], [H0], and [I0]);

100 mm in (H and I).

2 days in Myf5nlacZ/nlacZ embryos (Tajbakhsh et al., 1996),

whereas those in wild-type embryos migrate underneath the

DM and differentiate to form the myotome (anlage of trunk

muscles). However, Myod expression in somites eventually

rescues MPC fate from E11 and the developmental program

proceeds (Figures 3A and 3B; data not shown). Strikingly, EOM

progenitors in Myf5nlacZ/nlacZ embryos die rather than pause

(Figures 1B, 1D, 1I, 1I0, and 3D). We reasoned that EOM progen-

itors deficient for both Myf5 and Mrf4 are unable to progress in the

lineage. To analyze this, we coimmunostained E11.5 Myf5nlacZ/

nlacZ embryos for Myod, Myogenin, and b-gal. Interestingly, only

rare Myod- and Myogenin-positive cells were observed in the

EOM anlage of mutants, whereas in the control a large proportion

expressed these markers (Figures 3E–3F0, and 3G). In contrast,

MPCs in the first pharyngeal arch of mutants progress in the

lineage as efficiently as those in the control (Figures 3E, 3F,

3E00, 3F00, and 3G). Thus, while resumption of the myogenic

program follows Myod induction elsewhere in Myf5nlacZ/nlacZ

embryos, this process fails in EOM progenitors.

Interestingly, temporarily pausedsomitic MPCs of Myf5nlacZ/nlacZ

embryos do not apoptose, suggesting maintenance by other

survival factors. In somites, Pax3 is rapidly downregulated as

MRFs are expressed, but it is maintained in Myf5nlacZ/nlacZ

embryos suggesting that it is ensuring their survival (Tajbakhsh

812 Developmental Cell 16, 810–821, June 16, 2009 ª2009 Elsevier

et al., 1997). Pitx1 and Pitx2 are expressed in EOM progenitors,

and EOMs are lacking in Pitx2 null embryos, implicating this

gene in MPC survival and fate (Gage et al., 1999; Kitamura

et al., 1999). However, antibodies recognizing Pitx1/2 genes

continued to costain EOM progenitors in Myf5nlacZ as well as

Myf5nlacZ/nlacZ embryos. In spite of this expression, Pitx1/2 failed

to assure the survival of EOM progenitors in Myf5nlacZ/nlacZ

embryos (Figures 4A–4E). These results suggest that the MRFs

themselves, or another factor, are necessary for EOM survival.

In addition, MPC survival and cell identity or lineage progression

are intimately coupled.

Role of Myod in Head MusclesIf survival and lineage progression are coupled in MPCs, then

expression of Myod in embryos compromised for both Myf5

and Mrf4 should allow lineage progression and assure survival

of EOM progenitors as well. To test this hypothesis, we used

a knockin line, Myf5Myod-IRES-nlacZ/Myod-IRES-nlacZ, where Myod is

expressed from the Myf5 locus and Myf5 is knocked out. Notably,

Mrf4expression in thismutant is severely reduced, and lower than

that in Myf5GFP-P/GFP-P mutants (data not shown). Immunostain-

ing of E14.5 sagittal sections of Myf5Myod-IRES-nlacZ/Myod-IRES-nlacZ

embryos with anti-MyHC antibodies revealed that the EOMs were

rescued by Myod (Figure 4G). However, Myod, as noted for Mrf4,

Inc.

Developmental Cell

Distinct Regulatory Cascades Govern Head Myogenesis

Figure 2. EOM Progenitor Fate Is Determined by Myf5 or Mrf4

(A and B) Schemes identifying EOMs on whole mount ([A]; attached to eyeball; post-natal) and sagittal section ([B]; E14.5) in mouse. Sagittal sections of E14.5: (C)

wild-type; (D) Myf5loxP/loxP; (E) Myf5GFP-P/GFP-P; (F) Myf5nlacZ/nlacZ; (G) Myf5loxP/loxP:Mrf4nlacZ-P/nlacZ-P:Myf5nlacZ/nlacZ; and (H) Mrf4�/� immunostained for Myosin

heavy chain (MyHC). *, optic nerve provides anatomical landmark. Note that severity of phenotype corresponds to loss of both Myf5 and Mrf4; n = 3–4 embryos

for each mutant; EOMs demarcated by white boundary. (I) Scheme of EOMs attached to the eyeball, view from back with optic nerve pointing toward the viewer.

Whole mount preparations of EOMs attached to the eyeball, from adults of (J) wild-type, (K) Myf5loxP/loxP, and (L) Myf5GFP-P/GFP-P animals immunostained with

anti-MyHC antibody (n = 2–3 animals). Scale bars: 100 mm in (C–H).

does not appear to have a distinct antiapoptotic function in the

EOM progenitors, since in Myod�/�

animals EOM development

occured (Figure 4H). Thus, we conclude that EOM progenitor

survival as well as activation of Myod or Myogenin are dependent

on either Myf5 or Mrf4, and that any single MRF determination

factor is necessary and sufficient to assure MPC survival.

First Pharyngeal Arch Myogenesis Critically Dependson Tbx1 and Myf5

In the absence of Myf5 and Mrf4, induction of Myod in somitic

progenitors is brought about by Pax3. Surprisingly, we showed

that there is no factor which plays an equivalent role in EOM

progenitors. In contrast, Myod, and subsequently Myogenin,

are activated in the mesodermal core of the PAs in Myf5:Mrf4

double mutants (Figure 3F00 and 3G). Thus, another regulatory

pathway likely cooperates with Myf5 to activate Myod in PA

mesoderm. Pax7 is expressed in head MPCs and therefore it

Deve

may play a similar role to Pax3 in somitic myogenesis (Nathan

et al., 2008; Pownall et al., 2002; Sambasivan and Tajbakhsh,

2007). We generated Pax7nlacZ/nlacZ:Myf5GFP-P/GFP-P double

mutants and observed that PA-derived muscles were present

(Figures S2A and S2B) and the EOMs exhibited deficiencies

comparable to Myf5GFP-P/GFP-P fetuses (see Figure 2E). There-

fore, Pax7 is not the key regulator for establishing embryonic

MPC fate in these locations. Pitx2 is expressed in both EOM

and PA progenitors, but notably, Tbx1 which is required for robust

first arch myogenesis (Grifone et al., 2008; Kelly et al., 2004) is not

expressed in EOM progenitors (Dastjerdi et al., 2007). Therefore,

we investigated whether Tbx1 controlled the Pax3- and Myf5-

independent program in the head by examining PA muscles in

embryos null for both Tbx1 and Myf5. Interestingly, in situ hybrid-

ization (ISH) showed that Tcf21-expressing MPCs were bilater-

ally present in the first arch of E10. 5 Tbx1�/�:Myf5loxP/loxP

embryos (Figures 5A–5C, n = 2, embryos), as reported for

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Distinct Regulatory Cascades Govern Head Myogenesis

Tbx1�/�embryos (Kelly et al., 2004). In contrast, Myod transcripts

were bilaterally absent at E12.5 in spite of the presence of the an-

central myogenic cells in the PA mesodermal core (Figures 5D–F;

n = 4 embryos). To determine if this was not due to a delay in myo-

genesis, E15 Tbx1�/�: Myf5loxP/loxP and control fetuses were

immunostained for MyHC and sections at different levels were

compared to controls (Figures 5G–5L). In agreement with

a previous report (Kelly et al., 2004), Tbx1�/� mutants displayed

sporadic asymmetric first-arch-derived muscles (Figure 5J).

The PA muscle phenotype of Tbx1�/�: Myf5loxP/+ was similar, or

slightly more compromised than that of Tbx1�/� mutants

A B

C D

E F

E’

E’’ F’’

F’

G

Figure 3. Failure of Lineage Progression in

Extraocular MPCs Lacking Myf5 and Mrf4

(A and B) Somites and (C and D) EOMs, X-gal

stained Myf5nlacZ/+ (A and C) and Myf5nlacZ/nlacZ

(B and D) embryos; lateral view. Mrf4 is affected

in cis in Myf5nlacZ allele. White arrowheads point

to emerging pattern (C) and loss of MPCs in null

(D); white arrows mark EOM anlagen (C and D).

(E–F00) Immunostained sagittal sections of heads of

E11.5Myf5nlacZ/+ ([E], [E0], and[E00]) andMyf5nlacZ/nlacZ

([F], [F0 ], and [F00]) embryos, respectively. *, optic

nerve. (G) Quantification of Myod/Myogenin coex-

pressing cells in EOM anlagen and 1st PA core of

heterozygous (EOM, 42% ± 2.3 SEM; 1st PA, 50% ±

3.8 SEM; n = 3 embryos) and null mutants (EOM,

4% ± 0.5 SEM; 1st PA, 48.5% ± 4.3 SEM; n = 3

embryos each, 200-330 cells total; *p = 0.0092).

Scale bars: 100 mm in (E and F); 50 mm in (E0–F00).

(Figure 5K). Strikingly, first-arch-derived

muscles were missing in almost all

Tbx1�/�:Myf5loxP/loxP mutants analyzed

(Figure 5L). Although this phenotype is

incompletely penetrant (Table S1), overall

these results demonstrate that Myf5 is

required for the majority of the sporadic

myogenesis in the absence of Tbx1, and

that in the absence of both genes Myod

fails to program MPC fate.

In the body, Pax3 and Myf5, as well as Mrf4, operate upstream

of Myod. In Myf5 mutants where Mrf4 is active, most EOMs

develop; however, in Myf5loxP/loxP:Myod�/� mutants we

observed rare myoblasts expressing Desmin in the periocular

region, as well as in first PA (data not shown). This suggests

that Mrf4 alone cannot sustain myogenesis autonomously in

the absence of Myf5 and Myod in cranial mesoderm. As indi-

cated above, Mrf4 is expressed in EOM progenitors, but expres-

sion of the Mrf4nlacZ-P allele was not observed in pharyngeal arch

MPCs (n = 3; Figure S1F0–S1H00; data not shown). Therefore,

these observations suggest that in pharyngeal arch MPCs,

*

Myod-/-

wildtype

Pitx1β gal

Pitx2β gal

A

C D

EF

G

B

H

E14.5

**

MyHC Hoechst

posi

tive

cel

ls a

s %

of β

−gal

exp

ress

ing

cells

Pitx1 Pitx20

100

20

40

60

80

ControlMutant

Myf5nlacZ/+

Myf5nlacZ/nlacZ

Myf5Myod/Myod

Figure 4. Ectopic Myod Rescues Myf5

(Mrf4) Mutant EOM Phenotype

(A–D) Sagittal cryosections of E11.5 Myf5nlacZ/+

(A and C) and Myf5nlacZ/nlacZ (B and D) embryos

immunostained with anti-Pitx1 or Pitx2, and anti-

b-gal.

(E) Quantifications of coexpressing cells. Pitx1/

b-gal, 65.8% ± 1.2 in Myf5nlacZ/+; 64.2% ±

2.5 in Myf5nlacZ/nlacZ. Pitx2/b-gal, 96.1% ± 1.9 in

Myf5nlacZ/+; 94.8% ± 1.2 in Myf5nlacZ/nlacZ (n = 3

embyos each, 100–150 cells). Error bars, SEM.

Sagittal cryosections of E14.5, (F) wild-type, (G)

Myf5Myod-IRES-nlacZ/Myod-IRES-nlacZ, and (H) Myod�/�

fetuses stained with anti-MyHC antibody (n = 3

embryos each). See scheme in Figure 2B. Scale

bars: 50 mm in (A and D); 100 mm in (F–H).

814 Developmental Cell 16, 810–821, June 16, 2009 ª2009 Elsevier Inc.

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Distinct Regulatory Cascades Govern Head Myogenesis

1st PA

2nd PA1st PA

E F

Myo

d R

NA

ISH

E10.5 E10.5

E12.5 E12.5

3rd PA

Tbx1+/-

:Myf5loxP/loxP

Tbx1-/-

:Myf5loxP/loxP

Tbx1-/-

:Myf5loxP/loxP

Tbx1+/-

:Myf5loxP/+

Tbx1-/-:Myf5

loxP/loxPTbx1

-/- :Myf5

loxP/+

Tbx1 -/-

E15 E15

E15

E15

Wildtype

H

I J

Des

min

MyH

C

Hoe

chst

E15K L

Myf5loxP/loxP

CB

G

Tcf2

1 R

NA

ISH

SS

EOMsTongue

1st arch derived2nd arch derived

A

D

MasseterMylohyoid

Anterior digastric

1st PA2nd PA

FL

Figure 5. Tbx1 Cooperates with Myf5 to Initiate Pharyngeal Arch

Myogenesis

(A) Scheme of an E10.5 embryo head, boxed region shown in (B and C).

(B and C) Tcf21 mRNA in situ hybridization detects muscle progenitors in the

first arch of Tbx1:Myf5 double null (C) and control (B) embryos. Note, loss of

other arches was already reported in Tbx1 null embryos (Kelly et al., 2004).

(D) Scheme shows the region magnified in (E and F).

Deve

Tbx1 is required together with Myf5 to assure robust Myod

expression and myogenic cell fate.

Adult Head Muscle Satellite Cells Are Distinguishablefrom Satellite Cells of Somitic OriginAlthough satellite cells of head and trunk muscles have a distinct

origin (see Harel et al., 2009, in this issue of Developmental Cell),

all express Pax7. We asked if these adult satellite cells (SCs)

retain the ‘‘molecular memory’’ of their developmental history.

Adult head and hindlimb satellite cells were isolated by FACS

using transgenic Tg:Pax7-nGFP mice (Figure S3A) and expres-

sion of a set of genes implicated in head and trunk muscle

development was analyzed by quantitative RT-PCR. Interest-

ingly, we found that the SCs of EOMs and first PA-derived

muscles in the adult possess a molecular signature that is

distinct from their limb muscle counterparts (Figures 6A–6D).

Notably, Alx4 expressed in cranial mesoderm (Bothe and Die-

trich, 2006), and Pitx1 and Pitx2 are upregulated in EOM SCs

compared to limb SCs, whereas Pax3 and Lbx1 are not ex-

pressed. First-arch-derived SCs express high levels of Tcf21

compared to limb muscle SCs, but do not express Pax3. No

major differences in the expression of genes (eg., Pax7, Myf5,

Myod) common to all the programs were observed. Thus, these

findings show that SCs in different anatomical locations retain

distinct molecular signatures that reflect, in part, their develop-

mental history.

Fate of Heterotopically Transplanted EOM ProgenitorsIs Not Determined IntrinsicallyThe finding that the molecular signature of muscle stem/progen-

itor cells is distinct in different locations raises the question

whether this signature is hard-wired within these cells for auton-

omously conferring the distinct differentiated phenotypes of

various muscles. To address this issue, we performed hetero-

topic transplantations of SCs from adult transgenic mice

carrying three independent transgenes: (1) Tg:Pax7-nGFP

(nuclear GFP) that marks all satellite cells (Figure S3A; data

not shown); (2) Tg: MLC3F-nlacZ-2E that marks differentiated

myonuclei (Kelly et al., 1995); and (3) Tg: CAG-hPLAP (DePrimo

et al., 1996), a human placental alkaline phosphatase that is

expressed ubiquitously. We isolated by FACS and engrafted

EOM adult satellite cells into the Tibialis Anterior (TA) muscle

(E and F) In situ hybridization for Myod mRNA showing failure of Myod activa-

tion in first and second arch derived structures in E12.5 Tbx1:Myf5 (F)

compared to control (E). Both sides of the double null lacked or showed

severely reduced Myod mRNA (n = 4 embryos, not shown). PA, pharyngeal

arch; S, somitic; FL, fore limb.

(G) Scheme of sections for (H–L). Frontal cryosections of (H) wild-type, (I)

Myf5loxP/loxP, (J) Tbx1�/�; (K) Tbx1�/�: Myf5loxP/+; and (L) Tbx1�/�: Myf5loxP/loxP

E15 fetuses immunostained with anti-MyHC and anti-Desmin antibodies.

First-arch-derived muscles: masseter, mylohyoid, anterior digastric, ptery-

goid, and temporalis (Table S1) are missing in Tbx1�/�:Myf5loxP/loxP null

mutants. Arrows point to hypoplastic muscles in (K). Note, traces of green

staining in double mutants, also seen in controls, are nonspecific staining in

the mandibles. Loss of second-arch-derived muscles of facial expression (J)

has been reported in Tbx1 null embryos (Kelly et al., 2004). Owing to the plane

of sections, EOMs are not seen as much in (J) and (L) as in others. In 11/12

Tbx1�/�:Myf5loxP/loxP double mutants essentially all of these muscles were

lost (n = 8, E15; n = 4, E12.5; Table S1). Scale bars: 500 mm in (H–L).

lopmental Cell 16, 810–821, June 16, 2009 ª2009 Elsevier Inc. 815

Developmental Cell

Distinct Regulatory Cascades Govern Head Myogenesis

A C

D

F G

H

I’

I

B’ B’’

B

J

K L

E

Figure 6. Satellite Cells in Head Retain Their

Developmental Memory, but Fail to Confer

Original Muscle Phenotype in a Heterotopic

Location

(A) FACS scatter plot of EOM satellite cells (SCs)

from Tg:Pax7-nGFP.

(B–B00) Amplification plots of real-time RT-qPCR

with DRn (normalized fluorescence from ampli-

fying target) on y axis and number of amplification

cycles on x axis. Histograms of fold change (com-

paritive DDCT) in indicated transcripts between (C)

EOM and limb muscle SCs, or (D) pharyngeal arch

(PA) and limb muscle SCs. Alx4, Pitx1, and Pitx2

mRNAs were undetectable in PA SCs (data not

shown). Histogram represents mean fold change

of biological triplicates and technical duplicates;

error bars, SEM; *p < 0.05; ** < 0.005.

(E) Scheme of heterotopic transplantations of EOM

SCs.

(F) Cross-section of grafted TA muscle-immunos-

taining with anti-Laminin marks all myofibers;

placental alkaline phosphatase (PLAP) identifies

myofibers derived from donor EOM SCs.

(G) Cross-section of grafted TA muscle, immuno-

stained for sarcolemma marker a-sarcoglycan,

b-gal for donor EOM SC-derived differentiated

myonuclei (arrow heads; Tg: MLC3F-nlacZ-2E).

Arrows point to Tg:Pax7-nGFP positive b-gal

negative satellite cells.

(H) Cross-section of orbital layer of one of the adult

wild-type EOMs stained for Myh13, EOM-specific

Myosin heavy chain isoform.

(I and I0) Confocal images of nonengrafted (I) and

engrafted (I0 ) region marked by nlacZ positive

nuclei of the same muscle section stained for

Myh13. Images in ([H], [I], and [I0]) were acquired

with the same settings. Note that the region

grafted with EOM SCs ([I0], green) is negative for

Myh13.

(J) Differentiated culture (day 7) of EOM SCs iso-

lated by FACS.

(K and L) RT-qPCR fold difference in the expres-

sion of Myh13 and Myh15, between EOMs and

limb muscles, both in vivo (K), and between

EOMs (in vivo) and myotubes formed by EOM

SCs in vitro (L). Error bars, SEM. Scale bars:

100 mm in (F); 50 mm in (G–I0).

of Rag2�/�: gC�/� (Colucci et al., 1999) immunocompromised

recipient mice (Figure 6E). A robust engraftment efficiency was

achieved with large foci containing donor-derived myofibers

(Figure 6F; red). Engrafted cells also generated new SCs in the

host limb demonstrating that they can occupy new niches

(Figure 6G; green). However, myofibers generated from donor

EOM satellite cells failed to express markers of EOM muscles,

such as Myh13 (Figures 6H, 6I, 6I0, and S3D–S3G; Sartore

et al., 1987) and slow-tonic myosin (Figures S3B and S3C; Pe-

drosa-Domellof et al., 2000). Furthermore, nicotinamide dehy-

drogenase tetrazolium reductase diaphorase (NADH-TR) stain-

ing, reflecting the level of mitochondrial oxidative metabolism,

of donor-derived fibers (Fischer et al., 2005) was comparable

to that of surrounding nonengrafted TA muscle fibers and

much weaker than that of EOMs (data not shown). Taken

together, these results indicate that EOM satellite cells engraft

robustly in a heterotopic location by contributing to new fibers

816 Developmental Cell 16, 810–821, June 16, 2009 ª2009 Elsevier

and occupying niches, but they fail to program the EOM muscle

phenotype in this ectopic location.

To address this issue further, adult EOM satellite cells were iso-

lated by FACS, cultured in vitro, and allowed to differentiate.

Under these conditions, the potentially dominant influence of

a foreign muscle environment is avoided. Myotubes were then

isolated and analyzed by RT-qPCR for the presence of two

bona fide markers that distinguish between EOM and hindlimb

fibers (EOM specific myosins, Myh13 and Myh15; S. Schiaffino,

personal communication). Interestingly, EOM satellite cells failed

to generate EOM-type myotubes with the marker genes exam-

ined, even after 3 weeks of differentiation (Figures 6J–6L). As

a control, SCs from a slow muscle (Soleus) maintained their

slow phenotype after differentiation (Troponin T1 and Myh1;

data not shown). Taken together, these findings demonstrate

that the intrinsic genetic program of EOM satellite cells is insuffi-

cient to instruct the differentiated EOM phenotype autonomously.

Inc.

Developmental Cell

Distinct Regulatory Cascades Govern Head Myogenesis

DISCUSSION

Skeletal muscles have evolved the striking ability to adopt

selected elements of a core regulatory network in concert with

a complementary genetic pathway to assure self-renewal and

the acquisition of cell fate in different anatomical locations. These

pathways have been studied extensively in the body, yet the hier-

archical relationships of key regulators in cranial-derived MPCs,

and their relevance to the core MRF network, remained unre-

solved. We show that MPCs in the two major groups of head

muscles, the extraocular muscles and those in the pharyngeal

arches, have adopted surprisingly different modes of regulation

which are distinct from that governing MPC fate in the body.

Complementary studies using lineage tracing strategies show

that cranial satellite cells have a distinct origin from those in the

body (Harel et al., 2009, this issue). We show that skeletal muscle

stem cells in the adult retain their ontological molecular signature,

but these stem cells cannot autonomously assure the specific

muscle phenotypes in an ectopic location. These findings

provide a framework for investigating myopathies that are

restricted to subsets of muscles.

Initiation of EOM Cell Fate Is Governed by Myf5 or Mrf4

The myotome in the mouse, which is formed from the input of

MPCs that are born principally in the dorsal and ventral dermo-

myotome (DM) lips (Cossu et al., 1996; Tajbakhsh et al., 1996),

is a single undivided entity that patterns, splits, and displaces

into different muscle masses as this structure enlarges. Pharyn-

geal arch-derived and limb muscles follow a similar ontology.

Previous reports suggested that some EOM muscles in the chick

arise as independent anlage (Noden et al., 1999; von Scheven

et al., 2006). Observations with Myf5nlacZ animals suggest that

individual EOMs arise from a single, apparently undivided anlage

in mice, although this requires further investigation. We propose

that the EOM subsets are then patterned by local migrations of

progenitors away from this source to their more distal positions.

The mechanisms guiding the patterning process in the EOMs

remain to be explored.

Mrf4 drives the first embryonic wave of trunk myogenesis in

Myf5: Myod mutants (Kassar-Duchossoy et al., 2004), but its

role in head muscle development had not been elucidated. We

demonstrate that Mrf4 and Myf5 determine EOM progenitor

cell fate to form this distinct subset of head muscles of cranial

mesoderm origin. Apparently, both the prechordal and paraxial

head mesoderm contribute to EOMs in avians (Couly et al.,

1992; Noden and Francis-West, 2006; Wachtler et al., 1984).

Although data are lacking in the mouse concerning this point,

in either case, there is an obligate requirement for Myf5 or Mrf4

for initiating EOM fate.

Myod activation is mediated by Pax3, either directly or indi-

rectly, to initiate MPC fate in somites of Myf5:Mrf4 double

mutants. Interestingly, we show here that in the same mutants

neither Myod nor Myogenin rescue EOM development. Pitx2

acts upstream of MRFs in cranial MPCs and Pitx2 null embryos

lack EOMs (Gage et al., 1999; Kitamura et al., 1999); however,

the precise role of Pitx2 in inducing MRFs remained to be ad-

dressed. We show that Pitx2 fails to bring about the myogenic

rescue by Myod or Myogenin in EOM progenitors in the absence

of Myf5 and Mrf4, although some residual fibers were present in

Deve

these double mutants. Thus, Myod is epistatic to Myf5 and Mrf4

in EOM progenitors. We propose that unlike other skeletal

muscle progenitors (see below), EOM progenitors lack a Myf5/

Mrf4-independent pathway to activate myogenesis via Myod

(Figure 7).

Survival of Muscle Progenitors Is Coupledto CommitmentThe rapid apoptotic death of EOM progenitors in the absence of

Myf5 and Mrf4 is unusual among all MPCs in the embryo. In

Myf5:Mrf4:Myod triple mutants, some somitic MPCs also

undergo apoptosis, but several days after their birth (Kablar

et al., 2003; S.T., unpublished data). In both cases, MPCs fail

to commit and this may be linked to their death. Interestingly,

expression of Myod using a Myf5Myod allele, in the absence of

Myf5 and Mrf4, prevents the loss of EOM progenitors and

rescues EOMs. Thus, any one determination factor is sufficient

to ensure MPC survival. Other factors acting upstream may

also maintain MPC viability in the absence of MRFs. Interestingly

however, Myod plays a proapoptotic role in committed precur-

sors by inducing apoptosis in MPCs if they fail to differentiate,

and this requires p21/Cip1 (Peschiaroli et al., 2002). Additionally,

Myod null myoblasts are more viable than wild-type after trans-

plantion (Asakura et al., 2007). These opposing functions of the

MRFs need to be investigated in a developmental context.

Distinct Regulation of Pharyngeal Arch MPC Fateand Myogenic Cell PopulationsThe report that Pax3 activates somitic myogenesis through

a parallel program independent of Myf5 and Mrf4 (Tajbakhsh

et al., 1997) raised the possibility that another gene may do so

in cranial MPCs. We show that Pax7 in the head does not play

the early role of Pax3 in the body. The bilateral loss of first PA

muscles in Tbx1�/�:Myf5loxP/loxP double mutants suggests that

these genes are required together to initiate myogenic cell fate,

although Tbx1 may act genetically upstream of Myf5 as well

(Kelly et al., 2004). Apart from mesoderm, Tbx1 is expressed in

PA endoderm and ectoderm (Chapman et al., 1996) and hence

the analysis of the non-cell-autonomous role of this gene on

MPCs (Dastjerdi et al., 2007; Grifone and Kelly, 2007) needs to

be extended. Furthermore, unlike Pitx2 mutants, 2nd and 3rd

pharyngeal arches themselves are virtually absent in Tbx1 null

mutants, therefore the genetic hierarchy in the MPCs in these

arches remains an open question.

As reported for Pax3:Myf5(Mrf4) (Tajbakhsh et al., 1997) and

Msc:Tcf21 (MyoR:Capsulin, respectively) null mutants (Lu

et al., 2002), we note an incomplete penetrance and residual

myogenesis in some Tbx1�/�:Myf5loxP/loxP fetuses. This could

be due to compensation by Myod, another factor, or the genetic

background. Pitx2, expressed in the surface ectoderm and

mesodermal core of the first arch, is necessary for the specifica-

tion of the first arch itself, as the entire arch is severely reduced

in Pitx2 null mice (Dong et al., 2006; Shih et al., 2007). Elimi-

nating Pitx2 expression specifically in the mesoderm results in

a severe reduction of Myogenin expression (Dong et al.,

2006). Furthermore, Pitx2 and Tbx1 are known to regulate

each other (Nowotschin et al., 2006; Shih et al., 2007) and could

potentially act in parallel (Dong et al., 2006; Lamolet et al.,

2001). Therefore, we speculate that Pitx2 is a likely candidate

lopmental Cell 16, 810–821, June 16, 2009 ª2009 Elsevier Inc. 817

Developmental Cell

Distinct Regulatory Cascades Govern Head Myogenesis

for activating myogenesis in a smaller pool of arch MPCs in

Tbx1:Myf5 null mutants, resulting in some sporadic myogenesis

in the first arch. Taken together, we conclude that the combined

activities of Tbx1 and Myf5 are crucial for PA myogenesis

(Figure 7).

Our genetic analyses reveal that in EOM and PA MPCs, as in

somitic progenitors, Myod acts epistatically to Myf5. However,

on a cellular level, a Myf5-independent population was reported,

as identified by the failure of Myf5Cre to eliminate the entire MPC

pool after the conditional activation of a diptheria toxin gene

(Gensch et al., 2008; Haldar et al., 2008). Our data indicate that

a Myf5-independent population, if present in EOM, must be

largely dependent on Mrf4, or it is insignificant in EOMs. In the

first arch, we presume that this population would be largely

dependent on Tbx1, either in pharyngeal endoderm or meso-

derm, for Myod activation. We propose that the epistatic rela-

tionship between Myf5 and Myod is common for the majority

of muscle progenitors in the embryo. How cell nonautonomous

events could account for the rescue of myogenesis in the

absence of Myf5 and Mrf4 requires further investigation to deci-

pher the upstream decisions that decide which of the core MRFs

is selectively employed. Finally, we would speculate that two to

four genetic layers separate mesoderm formation from muscle

stem cell specification.

Figure 7. Core and Complementary Genetic

Networks Regulating EOM and PA Muscle

Fate

EOMs have dispensed with a complementary

regulatory pathway, therefore Myod expression

is not rescued in absence of Myf5 and Mrf4, and

EOMs do not form. In PA muscles, Tbx1 cooper-

ates with Myf5; in their combined absence, Myod

is not activated, and PA muscles are essentially

all missing. In the body (Kassar-Duchossoy et al.,

2004) and EOMs, Mrf4 determines embryonic

but not fetal MPC fate. Pitx2 may cooperate with

Tbx1 at this regulatory step in the PA. Pax3 acts

as the complementary pathway for body myogen-

esis to rescue Myod expression (Tajbakhsh et al.,

1997). With this model, the genetic distance

between mesoderm and muscle stem cells can

be envisioned. We speculate that two to four

layers separate these landmarks.

Adult MPC Molecular Signature andStem Cell Behavior in HeterotopicTransplantationsPax7 expressing stem/progenitor cells

appear in developing muscles throughout

the embryo around E11.5, after the estab-

lishment of a muscle anlage. A develop-

mental continuity exists between this

pool and adult SCs which are also depen-

dent on Pax7 for survival (Sambasivan

and Tajbakhsh, 2007). Hence, we were

surprised to observe that EOM and first-

arch-derived SCs bear distinct molecular

signatures compared to those in the

limbs. Remarkably, this distinct identity

partially reflects their developmental ontology. It would be inter-

esting to determine to what extent this occurs with stem cells in

other tissues.

Observations that skeletal muscles have specific functional

characteristics and differences in their ability to regenerate rai-

ses the question to what extent these phenotypes are conferred

by the stem/progenitor populations (Pavlath et al., 1998; Zammit

et al., 2006). Although heterotopic transplantation of temporalis

muscle of cat to the hind limb resulted in the regenerate

acquiring the donor jaw muscle identity (Hoy and Hughes,

1988), the cell autonomous role of SCs in conferring the donor

muscle phenotype remained unaddressed. Here we demon-

strate that SCs isolated directly from EOMs and transplanted

into a hind limb muscle engrafted robustly; however, EOM-

specific myosin expression was not observed in this ectopic

environment. In this context, we note that there are contradictory

reports regarding the intrinsic cue of limb muscle progenitors to

generate slow or fast fiber types. Whereas mouse fetal or post-

natal prequiescent myoblasts are not commited to form

a specific fiber type (Hughes and Blau, 1992; Robson and

Hughes, 1999), avian embryonic myoblasts appeared to be

restricted to slow or fast phenotypes (Nikovits et al., 2001).

Intriguingly, we report that the EOM SCs when differentiated

in vitro fail to express EOM-specific myosin heavy chain

818 Developmental Cell 16, 810–821, June 16, 2009 ª2009 Elsevier Inc.

Developmental Cell

Distinct Regulatory Cascades Govern Head Myogenesis

markers. Thus, we conclude that the EOM phenotype is not

dictated by the intrinsic cues of the stem/progenitors alone.

Trunk muscles function chiefly in locomotion and posture,

whereas the head muscles have varied functions such as eye

movements, feeding, and facial expression. These varied

requirements may have determined their distinct phenotypic

properties. We speculate that these properties reflect a develop-

mental underpinning. The distinct set of phenotypic characteris-

tics, in turn, is likely to underlie the differential involvement of

head muscles in a variety of myopathic diseases. Thus, under-

standing the differences in developmental programs and their

influence on the adult muscle phenotype is paramount to

building a framework to address the etiology of distinct

myopathic diseases.

EXPERIMENTAL PROCEDURES

Animals

Animals were handled as per European Community guidelines. Mouse lines

with mutant alleles, Myf5loxP, Myf5GFP-P, Myf5nlacZ, and Mrf4nlacZ:Myf5loxP,

were described previously (Kassar-Duchossoy et al., 2004; Tajbakhsh et al.,

1996). Myod�/� (Rudnicki et al., 1993); Mrf4�/� (kindly provided by E. Olson;

Zhang et al., 1995); Myf5Myod-IRES-nlacZ/Myod-IRES-nlacZ (provided by D. Gomes,

J. Perreau, G.D., D. Rocancourt, M. Buckingham, and S.T.); Tg: CAG-hPLAP

(courtesy of D. Daegelen; DePrimo et al., 1996); Tg: MLC3F-nlacZ-2E (Kelly

et al., 1995). For transplantations, Rag2�/�: gC�/� mice (Colucci et al., 1999)

were obtained from the Pasteur animal facilities. Mice carrying the Tbx1tm1pa

allele (referred to as Tbx1�) were genotyped as described; (Jerome and

Papaioannou, 2001). For generation of Tg: Pax7-nGFP line and Pax7nlacZ lines,

see the Supplemental Experimental Procedures. Mouse mutants were

genotyped and interbred as described (Kassar-Duchossoy et al., 2004).

In Situ RNA Hybridization, Immunofluorescence, and Imaging

ISH was performed as described (Kassar-Duchossoy et al., 2004; Tajbakhsh

et al., 1997) with Myod or Mrf4 riboprobes. Comparisons were done between

age-matched littermates hybridized under the same conditions. Images were

acquired with a Zeiss Axiocam numerical camera and Axiovision software.

Immunofluorescence was performed as described (Kassar-Duchossoy et al.,

2004). Embryos were fixed for 1.5 hr in 4% paraformaldehyde and embedded

in 7% gelatin and 15% sucrose embedded for cryosectioning (12–16 mm).

Dissected muscles were either 2% paraformaldehyde + 0.1% Tween20 fixed

for 1.5 hr or unfixed and cryofrozen in isopentane bath (�30�C) for sectioning

(12 mm). Sagittal sections were ideal for scoring all EOMs at E15 and frontal

sections were chosen for assessing pharyngeal arch-derived muscles. Images

were acquired using an Leica SPE confocal and LAS software or Zeiss Axio-

plan and Axiovision software. Optical sections (1–1.5 mm) were reconstructed

using ImageJ (NIH). Images were assembled using Adobe Photoshop and

Adobe InDesign. Antibodies used in this study include Myosin heavy chain

(rabbit, kindly provided by G. Cossu; 1/750), cleaved activated Caspase3

(mouse, Cell Signaling; 1/100 ; marker of apoptosis; Cohen, 1997), b-galacto-

sidase (rabbit, kindly provided by O. Puijalon; 1/200), Myod (Dako; M3512; 1/

50), Myogenin (DSHB; F5D; 1/10), Myh13 (DSHB; 4A6; 1/5; Lucas et al., 1995),

slow tonic myosin heavy chain (DSHB; S46; 1/10), placental alkaline phospha-

tase (GeneTex, Inc.; 73609; 1/400), Laminin (Sigma; 9393; 1/1000) GFP (Ab-

cam; 13970; 1/300), Pitx1, Pitx2 (polyclonal; kindly provided by J. Drouin),

and cyclin D1 (Thermo Scientific; SP4; 1/250 ; marker of cycling cells; Stacey,

2003). Tissue was not fixed for immunostaining with 4A6 and S46 antibodies.

Transplantation Experiments

Mice were anesthetized with 0.5% Imalgene/2% Rompun. Satellite cells iso-

lated by FACS, were injected into the Tibialis anterior muscle (3,000–10,000

cells) that had been subjected to three consecutive cycles of freezing-thawing

by applying a cryocooled metallic rod (Gayraud-Morel et al., 2007) 48 hr before

transplantation. Grafted muscles were collected 3 weeks posttransplantation

for analysis.

Dev

Quantitative RT-PCR

Total RNA was extracted from cells isolated by FACS using the Qiagen

RNAeasy Micropurification Kit. From 400–600 ng of DNase-treated (Roche)

RNA, cDNA was prepared by random-primed reverse transcription (Super-

Script II, Invitrogen) and real-time PCR done using powerSYBR Green

Universal Mix or Taqman universal Master Mix (ABI Prism 7700 and StepOne-

Plus, Perkin-Elmer Applied Biosystems). GAPDH transcript levels were used

for the normalizations of each target (= DCT). At least three biological repli-

cates were used for each condition (D(DCT) method) (Schmittgen and Livak,

2008). Custom primers were designed using the Primer3Plus online software

(http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Serial di-

lutions of total cDNA were used to calculate the amplification efficiency of

each primer set according to the equation: E = 10 � 1/slope. Primer dissocia-

tion experiments were performed to assure that no primer dimers or false am-

plicons would interfere with the results.

SUPPLEMENTAL DATA

Supplemental Data include three figures, two tables, and Supplemental Exper-

imental Procedures and can be found with this article online at http://www.cell.

com/developmental-cell/supplemental/S1534-5807(09)00210-X.

ACKNOWLEDGMENTS

We thank members of the lab and R. Grifone for helpful discussions; A. Jory for

help with establishing the Tbx1 colony; J. Drouin and P. Maire for reagents; M.

Nguyen-De Bernon for assistance with FACS, Pasteur Imaging platform

(PFID); F. Langa Vives and the Pasteur Transgenic Platform; D. Rocancourt,

M. Buckingham, E. Olson, S. Vincent, and D. Daegelen for mice; and S. Olivier

for artwork. Funding was granted by Institut Pasteur, AFM, FRM, ARC,

MyoRes (EU Framework 6), and EuroSyStem (EU Framework 7). R.K. is funded

by the AFM.

Received: December 21, 2008

Revised: April 9, 2009

Accepted: May 15, 2009

Published: June 15, 2009

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